1. Field of the Invention
The present invention generally relates to Limulus amebocyte lysate and more particularly to an improved method of removing suspected lipid inhibitors from the lysate.
2. Prior Art
Bacterial endotoxins are lipopolysaccharides associated with the outer membrane of gram negative bacteria. Various means for detecting the endotoxins have been devised. One of the newer test methods involves the use of lysate isolated from the amebocytes of the horseshoe crab, Limulus polyphemus. This method is very sensitive, that is, it can detect low level concentrations of the endotoxin in both aqueous samples such as pharmaceuticals and in human body fluids such as blood serum.
The lysate is obtained by osmotically bursting or otherwise breaking the cell wall of the amebocytes once they have been isolated from the blood of the crab, and separating out the cell wall debris. A positive test for the presence of Gram-negative bacterial infection (i.e. the presence of endotoxins in the blood sample) is gelation of the lysate within a reasonable amount of time after mixing it with the blood serum sample. Before this test is performed, the pH of the sample must be adjusted and any specific inhibitors to the gelation must be removed. Blood serum samples for instance must first be purified, as by chloroform extraction or the like, to remove gelation inhibitors from the blood. It has been found that, unfortunately, the sensitivity of the lysate seems to differ from batch to batch. It is now believed that certain natural lipids in the lysate are responsible for inhibiting or blocking the endotoxin gelation reaction. Removal of such lipid inhibitors increases the sensitivity of the lysate.
U.S. Pat. No. 4,107,077 to Sullivan and Watson deals with a method of removing such lipid inhibitors by extracting the lysate with a single organic solvent selected from the group consisting of chloroform, iodoform, bromoform, methyl bromide, methyl chloride, methyl iodide, ethyl chloride, ethyl bromide, ethyl iodide, propyl chloride, propyl bromide, propyl iodide, ethylene chloride, methylene chloride, chlorobenzene, bromobenzene, iodobenzene, dimethyl ether, diethyl ether, carbon tetrachloride, trichloroethylene, toluene and hexane. Chloroform is the solvent which this patent points to as having the greatest utility in this procedure. The procedure involves mixing the single solvent, such as chloroform, with two or more parts of the lysate, stirring gently for about an hour and then separating the lysate from the chloroform. While greatly improved lysate of greater uniformity is obtained by this procedure, unfortunately very little control is exercised by the procedure over the amount of protein in the lysate which is denatured, along with the inhibitor, by the solvent. Good, though not optimal, results are obtained but only with a very few solvents which exhibit a desirable degree of extractability.
It would be desirable to be able to provide an improved method of removing lipid inhibitors from Limulus amebocyte lysate which method could be optimized to denature the lipid inhibitors without adversely affecting the proteins in the lysate so as to improve the sensitivity and uniformity of the lysate to a greater degree than heretofore possible, regardless of the initial quality of the lysate. It would also be desirable to provide an improved method which would reduce the amount of solvent required to effect full and proper extraction and denaturation and/or reduce the total extraction and denaturation time.